This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Burkholderia pseudomallei is a category B potential bioterrorism agent, which causes melioidosis, a global emerging infectious disease. Thousands of cases of melioidosis have occurred throughout the tropics, and several cases have been reported in American travelers (civilian and military). B. pseudomallei infects host-cells through various stages: i) entry into host cell endocytic vesicles, ii) vesicular escape into the cytoplasm, iii) cytoplasmic replication, iv) utilize host actin for cytoplasmic mobility to v) catapult themselves into neighboring host-cells to spread the infection through membrane protrusions. We hypothesized that various genes are expressed to facilitate the transit of this bacterium through these different stages of cellular infection. Through gene-expression analyses, the long-term objective is to understand the molecular mechanism of pathogenesis of B. pseudomallei with respect to the intracellular environment of an infected cell. Two aims will identify essential genes and genes expressed on a spatiotemporal scale in a eukaryotic macrophage model. Aim 1 will identify genes expressed on a temporal scale during the various cellular infection processes. The expression of these genes will further be confirmed by reporter-gene-fusion and mutational analyses and video time-lapse recording. Aim 2 will employ a novel high-throughput negative-selection approach to identify genes essential for the infection process, which will be confirmed by real-time PCR, reporter-gene-fusion, and time-lapse recording of the infection process. We expect that achieving the objective will yield critical intervention strategies, novel drug targets, and vaccine candidates for the treatment and prevention of melioidosis.